Bottom Line:
Atomistic descriptions of the μ-opioid receptor (μOR) noncovalently binding with two of its prototypical morphinan agonists, morphine (MOP) and hydromorphone (HMP), are investigated using molecular dynamics (MD) simulations.Subtle differences between the binding modes and hydration properties of MOP and HMP emerge from the calculations.Comparison with an MD simulation of μOR covalently bound with the antagonist β-funaltrexamine hints to agonist-induced conformational changes associated with an early event of the receptor's activation: a shift of the transmembrane helix 6 relative to the transmembrane helix 3 and a consequent loss of the key R165-T279 interhelical hydrogen bond.

ABSTRACTAtomistic descriptions of the μ-opioid receptor (μOR) noncovalently binding with two of its prototypical morphinan agonists, morphine (MOP) and hydromorphone (HMP), are investigated using molecular dynamics (MD) simulations. Subtle differences between the binding modes and hydration properties of MOP and HMP emerge from the calculations. Alchemical free energy perturbation calculations show qualitative agreement with in vitro experiments performed in this work: indeed, the binding free energy difference between MOP and HMP computed by forward and backward alchemical transformation is 1.2±1.1 and 0.8±0.8 kcal/mol, respectively, to be compared with 0.4±0.3 kcal/mol from experiment. Comparison with an MD simulation of μOR covalently bound with the antagonist β-funaltrexamine hints to agonist-induced conformational changes associated with an early event of the receptor's activation: a shift of the transmembrane helix 6 relative to the transmembrane helix 3 and a consequent loss of the key R165-T279 interhelical hydrogen bond. This finding is consistent with a previous proposal suggesting that the R165-T279 hydrogen bond between these two helices indicates an inactive receptor conformation.

pone.0135998.g003: Representative structure of the MD simulations for (A) the MOP-μOR and (B) the HMP-μOR complexes obtained from clustering analysis.The ligand carbon atoms are in orange. H-bonds and salt-bridges are shown in green and magenta dashed lines, respectively. For clarity hydrogen atoms of the ligands and the μOR residues are not shown. H297 is monoprotonated at the Nε atom.

Mentions:
The two agonists (MOP and HMP) accommodate into the active site cavity in a slightly different manner, forming different H-bond patterns with residue H297 in TM6. In particular, MOP establishes a direct H-bond with H297 side chain, whereas HMP maintains water-mediated H-bonds with H297 side chain and K233 backbone mostly via one bridging water molecule (Fig 3 and S3 Fig). The presence of a cyclohexanone moiety within the HMP skeleton as opposed to a cyclohexenol ring, which is its cyclic counterpart in MOP, determines this different interaction pattern as it alters the drug surface available for hydrophobic interactions with protein residues in this region and slightly modifies the conformation of the polycyclic structure. MOP fits into the active site cavity establishing additional van der Waals contacts with G325, I296 and M151 while disrupting the initial interaction between D147 and the Y326 hydroxyl group. This disruption occurs at ~220 ns when Y326 moves away from D147 and eventually forms a very stable H-bond with T120. On the contrary, HMP binding causes the flip of the W293 side chain during the first 225 ns and gives rise to a stable interaction between the W293 and Y326 side chains (Fig 4). As a consequence, Y326 remains in close proximity to the ligand and preserves a H-bond with D147 (Fig 4B). The flip of the W293 side chain also leads to a rearrangement of the nearby aromatic residues, which establish different van der Waals contacts than those in MOP-μOR (Fig 4). The W293 reorientation separates HMP from G325 and places the ligand closer to I322, resulting in slightly different HMP-μOR hydrophobic contacts from those between MOP and the receptor (see Table 2). In addition, the flip of the W293 side chain allows more water molecules to enter the binding cavity below HMP (i.e. opposite to the extracellular side). After W293 flips, 15±2 water molecules are present inside the cavity of HMP-µOR, to be compared with 10±2 in MOP-μOR (S3 File). The different degrees of hydration are statistically significant, as indicated by normalized histograms and Welch’s t-test on the data (S3 File). Additional 0.5-μs MD simulations of MOP-μOR and HMP-μOR, starting from different initial velocities, reproduce the above-observed differences of the H-bond pattern, the W293 side chain orientation, the van der Waals contacts and the hydration in the binding cavity (S3 File).

pone.0135998.g003: Representative structure of the MD simulations for (A) the MOP-μOR and (B) the HMP-μOR complexes obtained from clustering analysis.The ligand carbon atoms are in orange. H-bonds and salt-bridges are shown in green and magenta dashed lines, respectively. For clarity hydrogen atoms of the ligands and the μOR residues are not shown. H297 is monoprotonated at the Nε atom.

Mentions:
The two agonists (MOP and HMP) accommodate into the active site cavity in a slightly different manner, forming different H-bond patterns with residue H297 in TM6. In particular, MOP establishes a direct H-bond with H297 side chain, whereas HMP maintains water-mediated H-bonds with H297 side chain and K233 backbone mostly via one bridging water molecule (Fig 3 and S3 Fig). The presence of a cyclohexanone moiety within the HMP skeleton as opposed to a cyclohexenol ring, which is its cyclic counterpart in MOP, determines this different interaction pattern as it alters the drug surface available for hydrophobic interactions with protein residues in this region and slightly modifies the conformation of the polycyclic structure. MOP fits into the active site cavity establishing additional van der Waals contacts with G325, I296 and M151 while disrupting the initial interaction between D147 and the Y326 hydroxyl group. This disruption occurs at ~220 ns when Y326 moves away from D147 and eventually forms a very stable H-bond with T120. On the contrary, HMP binding causes the flip of the W293 side chain during the first 225 ns and gives rise to a stable interaction between the W293 and Y326 side chains (Fig 4). As a consequence, Y326 remains in close proximity to the ligand and preserves a H-bond with D147 (Fig 4B). The flip of the W293 side chain also leads to a rearrangement of the nearby aromatic residues, which establish different van der Waals contacts than those in MOP-μOR (Fig 4). The W293 reorientation separates HMP from G325 and places the ligand closer to I322, resulting in slightly different HMP-μOR hydrophobic contacts from those between MOP and the receptor (see Table 2). In addition, the flip of the W293 side chain allows more water molecules to enter the binding cavity below HMP (i.e. opposite to the extracellular side). After W293 flips, 15±2 water molecules are present inside the cavity of HMP-µOR, to be compared with 10±2 in MOP-μOR (S3 File). The different degrees of hydration are statistically significant, as indicated by normalized histograms and Welch’s t-test on the data (S3 File). Additional 0.5-μs MD simulations of MOP-μOR and HMP-μOR, starting from different initial velocities, reproduce the above-observed differences of the H-bond pattern, the W293 side chain orientation, the van der Waals contacts and the hydration in the binding cavity (S3 File).

Bottom Line:
Atomistic descriptions of the μ-opioid receptor (μOR) noncovalently binding with two of its prototypical morphinan agonists, morphine (MOP) and hydromorphone (HMP), are investigated using molecular dynamics (MD) simulations.Subtle differences between the binding modes and hydration properties of MOP and HMP emerge from the calculations.Comparison with an MD simulation of μOR covalently bound with the antagonist β-funaltrexamine hints to agonist-induced conformational changes associated with an early event of the receptor's activation: a shift of the transmembrane helix 6 relative to the transmembrane helix 3 and a consequent loss of the key R165-T279 interhelical hydrogen bond.

ABSTRACTAtomistic descriptions of the μ-opioid receptor (μOR) noncovalently binding with two of its prototypical morphinan agonists, morphine (MOP) and hydromorphone (HMP), are investigated using molecular dynamics (MD) simulations. Subtle differences between the binding modes and hydration properties of MOP and HMP emerge from the calculations. Alchemical free energy perturbation calculations show qualitative agreement with in vitro experiments performed in this work: indeed, the binding free energy difference between MOP and HMP computed by forward and backward alchemical transformation is 1.2±1.1 and 0.8±0.8 kcal/mol, respectively, to be compared with 0.4±0.3 kcal/mol from experiment. Comparison with an MD simulation of μOR covalently bound with the antagonist β-funaltrexamine hints to agonist-induced conformational changes associated with an early event of the receptor's activation: a shift of the transmembrane helix 6 relative to the transmembrane helix 3 and a consequent loss of the key R165-T279 interhelical hydrogen bond. This finding is consistent with a previous proposal suggesting that the R165-T279 hydrogen bond between these two helices indicates an inactive receptor conformation.